Fossil fuel prices and environmental concerns have driven a search for renewable energy sources. Bioethanol is a type of biofuel that can be produced through the fermentation of biomass. First-generation bioethanol production utilizes sugar and starch from crops as a feedstock. It encounters multiple challenges such as rising food prices and scarcity of arable farmland, resulting in an urgent need for alternative solutions. Third-generation bioethanol production uses algae and requires the biomass to be pretreated and hydrolyzed for saccharification as well as generation of fermentable sugars to be used by other microorganisms in order to produce bioethanol through fermentation. However, pretreatment and hydrolysis of biomass are often accompanied by the generation of inhibitors, which seriously impede subsequent microbial fermentation to produce bioethanol. Thus, the key challenges associated with this energy source are related to the ability to produce bioethanol more efficiently without competing for food crop supply and cultivable land.
Immobilization is a way to isolate or localize intact cells in a certain space and maintain their catalytic activity. Immobilized cells can effectively reduce the negative effects of inhibitors and the processing cost of inoculum preparation for continuous or fed-batch fermentation of microorganisms. Immobilization techniques can be categorized as follows: (1) immobilization on solid carrier surfaces, (2) entrapment within a porous matrix, (3) mechanical containment behind barriers, and (4) cell flocculation (aggregation). Bioethanol fermentation using immobilized cells can increase cell density, shorten fermentation time, increase ethanol and inhibitor tolerance, and improve the feasibility of using continuous fermentation, resulting in more efficient bioethanol production. When sodium alginate beads were used for immobilization with Saccharomyces cerevisiae for bioethanol fermentation, it was found that immobilized yeast cells converted glucose into alcohol more efficiently than free yeast cells during batch fermentation, and the beads immobilized with yeast cells could be reused for 5 consecutive batch runs. Moreover, using multihole sterile loofah sponges or cicada cocoons for Kluyveromyces marxianus immobilization enhanced ethanol fermentation compared with that using free cells. Furthermore, S. cerevisiae VS3 immobilized on rice straw boosted bioethanol fermentation and was reusable for 8 consecutive batch runs (A. K. Chandel, M. L. Narasu, G. Chandrasekhar, A. Manikyam, L. V. Rao, Use of Saccharum spontaneum (wild sugarcane) as biomaterial for cell immobilization and modulated ethanol production by thermotolerant Saccharomyces cerevisiae VS3. Bioresour. Technol. 100 (2009) 2404-2410).
The current techniques to immobilize cells have several drawbacks. The surface attachment of cells using chemical linking agents, such as glutaraldehyde, may be unsuitable for the production of ethanol or beverages. Moreover, because there are no barriers between solution and cells, cell detachment and relocation may contaminate products. Another approach to cell immobilization is using a porous gel matrix, such as Ca-alginate, to entrap cells and obtain high biomass loadings for fermentation; however, the bead structure can destabilize in the presence of acid or diffusion limited gases, such as CO2 in ethanol fermentation, and result in bead rupture. Cell flocculation is considered a relatively low-cost method; however, the flocculation of some yeasts is inhibited by the presence of sugars, such as glucose, and ethanol. Using the method of containment behind a barrier, such as that used in microporous membrane filters, for cell immobilization is most suitable when a cell-free product is required. However, there are inherent problems such as possible membrane fouling, high cost, and container recycling issues (D. J. O'Brien, L. H. Roth, A. J. McAloon, Ethanol production by continuous fermentation-pervaporation: a preliminary economic analysis, J. Membr. Sci. 166 (2000) 105-111).